scholarly journals Whole Genome Sequencing of B-Cell Neoplasms with t(14;19)(q32;q13) Reveals Different Entities

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3709-3709
Author(s):  
Anna Carbó-Meix ◽  
Francesca Guijarro ◽  
Luojun Wang ◽  
Romina Royo ◽  
Isabel Granada ◽  
...  
Keyword(s):  
B Cell ◽  
Base Pair ◽  
Research Funding ◽  
Differential Expression Analysis ◽  
Lymphocytic Leukemia ◽  
Whole Genome ◽  
Rna Seq ◽  
Molecular Features ◽  
Genomic Complexity ◽  
Mutational Status

Abstract Introduction: The t(14;19)(q32;q13) is a rare cytogenetic abnormality found in <0.1% of all B-cell neoplasms. The molecular features of this translocation are not well characterized. IGH-BCL3 rearrangement has been found in some tumors identified as "atypical" chronic lymphocytic leukemia (CLL) with aggressive clinical evolution. This translocation has also been observed in other B-cell neoplasms without clear evidence of the target gene. The mechanisms generating this translocation, the genomic profile of alterations of these cases, and whether different molecular features may be associated with specific entities are not known. Aim: To elucidate the genomic features of B-cell neoplasms carrying the t(14;19) and their relationship to pathological characteristics of the tumors. Materials and methods: We sequenced the whole-genome (WGS) of 13 cases in which the t(14;19) had been identified by conventional cytogenetics and/or FISH using a BCL3 break-apart probe. In six of these cases we performed RNA-seq. Pathological and clinical revision was conducted in all cases, 8 of them with tissue biopsies. Results: The breakpoints of the t(14;19) were characterized at base-pair resolution using WGS. All breakpoints in chr14 were found within any of the class switch recombination (CSR) regions suggesting an aberrant CSR as the mechanism causing this alteration. The breakpoints on chr19 were found upstream (13 kb) the 5' untranslated region (UTR) of BCL3 in 8/13 (61.5%) cases. One additional case had the breakpoint further upstream (49 kb) of BCL3 truncating CEACAM16. The four remaining cases had breakpoints downstream of BCL3; two cases within CBLC, one in BCAM, and one after NECTIN2. Of note, the further upstream BCL3 case and the downstream BCL3 cases had mutated IGHV, while all upstream BCL3 cases had unmutated IGHV. Based on RNA-seq data, all upstream BCL3 cases (n=5) showed an upregulation of BCL3, while one downstream case with RNA-seq available showed upregulation of NECTIN2 and low levels of BCL3. The pathology review identified the four downstream BCL3 cases as marginal zone lymphomas whereas the cases with breakpoints upstream BCL3 (n=3 with tissue available) and the case further upstream BCL3 were classified as "atypical" CLL. We next characterized the genomic landscape of these tumors based on the breakpoint on chr19 (upstream and downstream BCL3). The analysis of the WGS showed a lower number of mutations, copy number alterations (CNA), and structural variants (SV) in the upstream BCL3 group compared to the downstream BCL3 cases (mean of 2429.5 vs 6271.7 somatic mutations, 3.1 vs 11.7 CNA, and 4.4 vs 18 SV, respectively). In terms of specific driver mutations, the downstream BCL3 group carried mutations in genes previously described in MZL, such as KMT2D, NOTCH2, or KLF2 found in two cases. All but one case with the breakpoint upstream BCL3 carried trisomy 12 (tri12), which was absent in all cases with a downstream breakpoint. Finally, we performed a differential expression analysis between 5 atypical CLL cases with BCL3 rearrangements vs 4 CLL without t(14;19) [all unmutated IGHV]. This analysis showed 578 genes upregulated and 720 genes downregulated in the BCL3-rearranged cases (q <0.05), including remarkable differences in the expression of previously described CLL hallmark genes, such as upregulation of EBF1 and downregulation of LEF1, FMOD, ADTRP, CLNK, IGSF3, TCF4. An analysis of the RNA-seq data of 294 CLL cases lacking the t(14;19) (Puente et al., Nature 2015) indicated that this transcriptional program was not related to IGHV mutational status nor to the presence of tri12. Nonetheless, we identified a small set of tri12 mutated IGHV CLL lacking the t(14;19) with a similar modulation of the expression of the above hallmark genes. Conclusions: We have characterized the breakpoints of the t(14;19) at base-pair resolution and evidenced marked molecular and pathological differences of the tumors according to the location of the breakpoint. Tumors carrying the breakpoint downstream BCL3 exhibit a higher genomic complexity, driver alterations, and pathological features corresponding to MZL. Contrarily, tumors with the breakpoint upstream of BCL3 upregulate BCL3 and display lower genomic complexity as well as CLL-like features. Nonetheless, these cases have a different gene expression profile compared to conventional CLL characterized by LEF1 downregulation and EBF1 overexpression. Disclosures Navarro: Nocartis: Honoraria; Roche: Honoraria; EUSA: Consultancy, Research Funding; Pharma: Consultancy; GILEAD: Research Funding; Pharma: Research Funding.

scholarly journals The U1 Spliceosomal RNA: A Novel Non-Coding Hotspot Driver Mutation Independently Associated with Clinical Outcome in Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 847-847
Author(s):  
Ferran Nadeu ◽  
Shimin Shuai ◽  
Ander Diaz-Navarro ◽  
Irene López ◽  
Silvia Martín ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Expression Patterns ◽  
Lymphocytic Leukemia ◽  
Small Nuclear Rna ◽  
Rna Seq ◽  
Wild Type ◽  
Base Pairing ◽  
Mutational Status

Introduction: Genomic studies of chronic lymphocytic leukemia (CLL) have uncovered >80 potential driver mutations. The vast majority of these mutations affect coding regions, and just two potential drivers have been identified in non-coding elements. Aim: To describe the biological and clinical impact of a recurrent A>C mutation at the third base of the small nuclear RNA U1, the non-coding component of the spliceosome involved in the recognition of the 5' splice site (5'SS). Methods: Whole-genome sequencing (WGS) and RNA-seq from 318 CLL patients were used to identify and characterize a highly recurrent A>C point mutation occurring at position 3 of the U1 snRNA gene (g.3A>C mutation). The U1 wild-type and mutant forms were introduced into three CLL cell lines (JVM3, HG3, MEC1) to validate in vitro the predicted effect of this alteration. We screened two independent cohorts including a total of 1,314 CLL patients for the presence of the mutation using the rhAmp SNP genotyping assay, and integrated the U1 mutational status with well-known driver alterations, IGHV and epigenetic subgroups, and clinical parameters. Results: The U1 mutation was found in 8/78 (10.3%) CLL cases analyzed by WGS. Given its role in 5'SS recognition by base-pairing, we reasoned that this mutation was likely to alter the splicing and expression patterns of CLL. We were able to confirm widespread specific alterations in the transcriptome by comparing RNA-seq data between wild-type and g.3A>C mutated samples. Applying this knowledge to an algorithm aimed to infer the U1 mutational status from expression data, we were able to identify 4 mutated cases among 240 additional cases that had RNA-seq but no WGS. In total, 12/318 (3.8%) CLL patients analyzed by WGS and/or RNA-seq harbored this mutation. This g.3A>C U1 mutation changes the preferential A-U base-pairing between U1 and 5'SS to C-G base-pairing, creating novel splice junctions and altering the splicing pattern of 3,193 introns in 1,519 genes. In addition to altered splicing, 869 genes were differentially expressed between mutated and wild-type cases. We identified specific cancer genes (e.g. MSI2, POLD1, or CD44) and pathways (B-cell receptor signaling, promotion of apoptosis, telomere maintenance, among others) altered by the U1 mutation. To confirm a causal link between this mutation and splicing changes, we introduced exogenous U1 genes with or without the mutation into three cell lines. Subsequent RNA-seq of these cell lines recapitulated the altered splicing and expression patterns observed in CLL patients. We next screened for the presence of the U1 mutation 1,057 patients (cohort 1) using the rhAmp assay and it was found in 30 (2.8%) cases. The distribution of the mutation was similar in Binet stages and CLL vs monoclonal B-cell lymphocytosis. However, the U1 mutation was almost always found in IGHV unmutated CLL (29/30, p=9.0e-11) and within the naïve-like CLL epigenetic subgroup (p=3.7e-7). None of the U1 mutated cases had mutations in the SF3B1 splicing factor. Considering only pre-treatment CLL samples, U1 mutation was associated with a shorter time to first treatment independently of the Binet stage, IGHV mutational status, epigenetic subgroups, and mutations in the well-known CLL drivers SF3B1, NOTCH1, ATMor TP53. In cohort 2 (n=257), this mutation was found in 13 (5.1%) patients, confirming its enrichment in IGHV unmutated cases, naïve-like epigenetic subgroup, and splicing modulation. Despite the relatively small number of pre-treatment samples carrying the U1 mutation (7/178) and short follow-up of the patients (median 2.6 years), the effect of this mutation on time to first treatment in cohort 2 was compatible with the one observed in cohort 1. Finally, we screened for the U1 mutation a cohort of diffuse large B-cell lymphoma (n=108), mantle cell lymphoma (n=101), follicular lymphoma (n=87), splenic marginal zone lymphoma (n=12), acute myeloid leukemia (n=52), and myelodysplastic syndrome (n=67). The mutation was not present in any of the samples analyzed. Conclusions: Here we have reported that the third base of the small nuclear RNA U1 is recurrently mutated in CLL, proved its effect in splicing and gene expression, and shown that this mutation is independently associated with faster disease progression. The g.3A>C U1 mutation represents a novel non-coding driver alteration in CLL with potential clinical and therapeutic implications. Disclosures Ramirez Payer: GILEAD SCIENCES: Research Funding. Terol:Astra Zeneca: Consultancy; Gilead: Research Funding; Abbvie: Consultancy; Janssen: Consultancy, Research Funding; Roche: Consultancy. Lopez-Guillermo:Celgene: Consultancy, Research Funding; Janssen: Research Funding; Roche: Consultancy, Research Funding; Gilead: Consultancy, Research Funding.

scholarly journals SYK Inhibition Disrupts the Cross-Talk Between B-Cell Activation Factor (BAFF) and B-Cell Receptor (BCR) and Thereby Antagonizes Mcl-1 in Chronic Lymphocytic Leukemia (CLL) B-Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 303-303
Author(s):  
Cody Paiva ◽  
Taylor Rowland ◽  
Olga Danilova ◽  
Bhargava Sreekantham ◽  
Stephen E Spurgeon ◽  
...  
Keyword(s):  
B Cells ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Cell Survival ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Lymphocytic Leukemia ◽  
Mutational Status ◽  
Pathway Gene ◽  
Bcr Signaling

Abstract Although small molecule inhibitors of BCR-associated kinases (BCRi) revolutionized therapy in CLL, they provide incomplete responses. Soluble mediators emanating from the tumor microenvironment perpetrate CLL cell survival and may account for resistance to BCRi. Tumor necrosis factor receptor superfamily ligands BAFF and APRIL induce NFκB, which in turn upregulates pro-survival Bcl-2 family proteins and thereby drives anti-apoptotic responses.The exact roles of the individual NFκB pathways, as well as the implications of targeting BCR in context of BAFF signaling in CLL remain understudied. We explored the mechanistic underpinnings of CLL cell survival in response to BAFF signaling, uncovering the functional significance of the BCR-associated kinases and Bcl-2 family proteins in this setting. Peripheral blood mononuclear cells were isolated from patients with CLL. We established a novel BAFF-expressing stromal co-culture model and referenced it to control, CD40L-expressing stroma and soluble BAFF. We employed inhibitors of Bruton tyrosine kinase (BTK, ibrutinib), phosphoinositide-3 kinase (PI3K, idelalisib) and spleen tyrosine kinase (SYK, entospletinib) and measured CLL cell apoptosis, migration, NFκB activity, protein and mRNA expression by flow cytometry, immunoblotting, ELISA, RT-PCR and immunocytochemistry. CLL cells co-cultured with BAFF-expressing stroma were resistant to spontaneous apoptosis (12.3±3.2% after 24 h, vs 34.8±6.2% off stroma) and chemotherapy agents (bendamustine, fludarabine). Gene expression profiling exposed the NFκB pathway gene targets as the most significantly upregulated upon BAFF stimulation (p<0.0001). We and others have shown that CD40L-expressing stroma induces canonical and non-canonical NFκB in CLL. By contrast, while BAFF led to strong activation of the non-canonical NFκB with processing of p100 (to p52) by 4 h and a 5-fold increase in p52 DNA-binding activity by 24 h, canonical NFκB (RelA) activation was less pronounced. BAFF predominantly induced Mcl-1, compared to CD40L which strongly upregulated Bcl-X. BCR is a major driver of canonical NFκB signaling in CLL. Thus, we studied whether BAFF co-opted BCR signaling in CLL. BAFF induced rapid (15 min) phosphorylation of the proximal BCR kinases SYKand LYN, sustained for up to 4 h, as well as ERK, in CLL cells. AKT activation occurred late (>2 h), suggesting that BAFF induced AKT independent of BCR. BAFF-mediated BCR activation did not correlate with IGHV mutational status. Like IgM, BAFF induced CLL cell chemotaxis. SYK inhibition effectively antagonized survival and chemotaxis of BAFF-stimulated CLL cells. By contrast, targeting BTK or PI3K was less effective. All BCRi's fully blocked canonical NFκB activation in BAFF-stimulated CLL cells (suggesting its dependence on BCR signaling), but none inhibited the non-canonical pathway. We found that entospletinib, but not other BCRi's, decreased Mcl-1 expression in CLL cells co-cultured with BAFF-expressing stroma. Unlike in IgM-stimulated cells, entospletinib did not promote Mcl-1 protein degradation. By contrast,, targeting SYK in BAFF-stimulated cells abrogated BAFF-mediated upregulation of pSTAT3, a transcription factor which regulates Mcl-1. This was accompanied by a decrease in Mcl-1 transcript, an effect mimicked by ruxolitinib, a JAK/STAT inhibitor. BAFF receptor signals via the TRAF3/NIK/IKK1 axis to induce non-canonical NFκB activation in neoplastic B-cells. We supposed that NIK (NFκB-inducing kinase) or IKK1 could be directly responsible for SYK activation by BAFF. Indeed, genetic knockdown of NIK resulted in decreased SYK activation, whereas IP experiments demonstrated that NIK directly complexed with SYK in BAFF-stimulated neoplastic B-cells, confirming NIK role in activation of BCR signaling. Thus, BAFF-mediated induction of BCR-associated kinases and Mcl-1 contributes to CLL cell survival. SYK inhibition is a promising therapeutic strategy uniquely poised to antagonize crosstalk between BAFF and BCR, thereby disrupting the pro-survival microenvironment signaling in CLL. Disclosures Spurgeon: Gilead Sciences: Research Funding; Bristol Myers Squibb: Research Funding; Acerta Pharma: Research Funding; Genentech: Research Funding; Janssen: Research Funding. Danilov:Prime Oncology: Honoraria; Dava Oncology: Honoraria; ImmunoGen: Consultancy; GIlead Sciences: Research Funding; Takeda: Research Funding; Astra Zeneca: Research Funding; Pharmacyclics: Consultancy.

Array-CGH Based Genomic Profiling in B-Cell Chronic Lymphocytic Leukemia Reveals Specific Correlations of Genomic Imbalances and Prognostic Subgroups and Underlines the Consistency of Chromosomal Aberration Patterns within This Disease.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2083-2083
Author(s):  
Carsten Schwaenen ◽  
Dirk Kienle ◽  
Alexander Krober ◽  
Sandra Ruf ◽  
Dirk Winkler ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Chromosomal Aberration ◽  
Chromosomal Aberrations ◽  
Lymphocytic Leukemia ◽  
Genomic Profiling ◽  
Genomic Imbalances ◽  
Genomic Complexity ◽  
Mutational Status ◽  
Genomic Aberrations

Abstract Chronic lymphocytic leukemia of B-cell type (B-CLL) is characterized by a number of typical genomic aberrations. In comparison to patients with normal karyotypes or 13q deletions patients with high risk imbalances such as deletions of 11q, 17p or unmutated IgVH status have a higher risk for advanced disease and a significantly shorter survival. For a precise mapping of chromosomal imbalances as well as to verify the number of aberrations per case in different genetic subgroups of B-CLL (e.g. del11q, del13q, del17p, +12 or unmutated IgVH status) we performed high resolution genomic profiling using a genomic DNA-chip containing 2.800 probes. Target clones compriseda large genome-wide cluster of clones covering the genome at a distance of approx. 1.5Mb andclones mapping to genomic regions or genes of possible pathogenetic relevance in lymphoma. This chip covers approximately 10% of the human genome. In 93 (70%) of 133 analyzed B-CLL cases 171 genomic imbalances were identified (between 1–7 aberrations/case). Besides the confirmation of known recurrent chromosomal aberrations, previously unknown recurrent imbalances were detectable on 2p (8%), 4p (4%), 7p-q (5%), 10q (5%) and 20p (3%). Most of these imbalances were of larger extension (> 10 Mb) and therefore impeded a further delineation of minimal aberrant regions and the identification of possible candidate genes. The mean number of chromosomal aberrations per case (= genomic complexity) in IgVH unmutated CLLs was approx. 2 times higher than in mutated cases (0,77 vs. 1,58 per case). 84% of samples with > 2 aberrations showed an unmutated IgVH status. Moreover, most of the previously unknown imbalances were identified within this group. A higher genomic complexity was also shown for samples with gain on 2p vs. balanced 2p status (2.2 times higher; 2.5 vs. 1.2) and in samples with del17p vs. balanced 17p status (3.7 times higher; 3.52 vs. 0.95). 11q aberrations had no impact on the number of genomic aberrations per case (1.6 vs. 1.2). Moreover, we found a strong association of 2p gains and an unmutated IgVH status (100%). Array based genomic profiling confirmes the chromosomal aberration structure and underlines the consistency of chromosomal aberration patterns of B-CLL. The biological and prognostic relevance of 2p gains and unmutated IgVH mutational status have to be further investigated.

scholarly journals Systematic Evaluation of Somatic Cis-Regulatory Mutations in Follicular Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 26-27
Author(s):  
Firat Uyulur ◽  
Findlay Bewicke-Copley ◽  
Chinedu Anthony Anene ◽  
Matthias Schlesner ◽  
Icgc MMML-Seq Project ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Mutation Rate ◽  
Research Funding ◽  
Whole Genome ◽  
Rna Seq ◽  
Regulatory Mutations ◽  
Flanking Sequences ◽  
Bona Fide ◽  
Flanking Regions

Background: Follicular lymphoma (FL) is an incurable indolent B cell malignancy characterized in the majority of cases by the t(14;18) translocation. While the mutational landscape of the coding genome is nearing completion, less is known about the characteristics of its noncoding genome. Our expectation is that the distribution of noncoding mutations will be non-random, determined by epigenomics features, such as chromatin modification and accessibility. Our strategy is therefore to integrate whole genome epigenomic marks and mutations in order to enrich for variants with regulatory potential and identify unique mutational processes specific to thesecis-regulatory elements (CREs).Allele-specific expression (ASE) patterns allow further refinement to resolve functional CREs and bona fide mutations associated with changes in gene expression. Methods: The H3K27Ac consensus CREs were determined using the ChIP-Seq data of 9 FL patient primary cells (Koues et al., Immunity2015). DNase I-hypersensitive sites (DHSs) and Hi-C of the B-lymphocyte cell line GM12878 was downloaded from ENCODE. Whole genome sequencing and RNA-Seq of 70 FL patients with relative high tumour cellularity (≥30% for DNA tumour purity and ≥25% for RNA B-cell content) were obtained from the International Cancer Genome Consortium project ICGC MMML-Seq. The mutation rate within CREs and DHSs in comparison to their flanking sequences was estimated using a previous pipeline (Sabarinathan et al., Nature2016). We further developed cis-ASE, an integrated analytic pipeline for the identification of recurrent ASE genes, and significantly associated CREs and mutations (Fig1.A). Results: In total 1.04 million noncoding mutations, corresponding to 14.8K mutations per sample with a median of 9,991 noncoding mutations were identified in our series of 70 FL samples. 62K (6.0%) mutations were located within H3K27Ac bound CREs and there was an elevated mutation rate in H3K27Ac CREs compared to the corresponding flanking regions of 1kb up and downstream (χ² test p = 1.95e-19, Fig1.B). For DHSs, we observed the opposite pattern, with a lower mutation rate in DHSs than in the flanking regions (p = 0.04, Fig1.C), most likely reflecting the higher accessibility to global genome repair machinery in DHSs in relation to flanking sequences. Dividing mutations into high (≥0.3) and low (&lt;0.3) adjusted variant allele frequency (VAF) groups (accounting for tumour purity), we observed significantly higher mutation rate in CREs than in the flanking sequences, that was specific for mutations with higher VAFs (p= 6.95e−38), as the difference was much weaker for low VAF mutations (p= 0.02). Mutation signatures 6 and 20, linked to defective DNA mismatch repair, were highly enriched for mutations within H3K27Ac CREs (44%) versus other regions outside (27%) (p&lt; 0.001). ASE was assessed using 45.6k informative SNPs (1.7%) per sample wherein matched genotype data was available from WGS and RNA-Seq profiles. Cis-ASE identified on average 480 ASE genes per sample (binomial test, adjusted p&lt;0.05), corresponding to 1,943 recurrent ASE genes with a minimum threshold of ≥5 samples. These ASE events were not significantly influenced by local copy number changes or promoter methylation. KEGG pathway analysis of recurrent ASE genes identified adherens junction, B cell receptor signaling pathway and Fc gamma R-mediated phagocytosis as the most overrepresented pathways. Cis-ASE further identified 18 ASE-CRE interactions where CRE mutations were significantly correlated with an imbalance in elevated alternative allele ratios. These ASE genes included recognized lymphoma related genes including BCL2, STAT6, MAF, and additional novel targets. The pattern of aberrant somatic hypermutation (aSHM) was assessed for these 18 CREs, and we narrowed down to 15 significant ASE-CRE interactions not strongly affected by aSHM, consisting of 92 bona fide mutation candidates present in 37 FL samples of our cohort (52.9%). Conclusion: Our study identified unique mutation processes operating in H3K27Ac CREs. Using an integrated genomic approach of whole genome mutations, chromatin marks and RNA-Seq, we explored ASE-CRE interactions, and identified 15 H3K27Ac bound CREs enriched for cis-regulatory mutations significantly associated with ASE and total expression of targeted genes, deserving for further exploration to enrich our understanding of FL noncoding genome. Disclosures Okosun: BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences: Research Funding.

scholarly journals Relation of Gene Expression Phenotype to Immunoglobulin Mutation Genotype in B Cell Chronic Lymphocytic Leukemia

2001 ◽  
Vol 194 (11) ◽  
pp. 1639-1648 ◽  
Author(s):  
Andreas Rosenwald ◽  
Ash A. Alizadeh ◽  
George Widhopf ◽  
Richard Simon ◽  
R. Eric Davis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Germ Line ◽  
Gene Expression Signature ◽  
Lymphocytic Leukemia ◽  
Common Mechanism ◽  
Human Leukemia ◽  
Mutational Status ◽  
Cell Chronic Lymphocytic Leukemia

The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression “signature,” irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.

Distinct Gene Expression Signatures in Patients with Richter's Syndrome and Chronic Lymphocytic Leukemia with Prior Exposure to Ibrutinib

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-31
Author(s):  
Hanyin Wang ◽  
Shulan Tian ◽  
Qing Zhao ◽  
Wendy Blumenschein ◽  
Jennifer H. Yearley ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Activation ◽  
Expression Profiles ◽  
Lymphocytic Leukemia ◽  
B Cell Activation ◽  
Advisory Committees ◽  
Upregulated Genes

Introduction: Richter's syndrome (RS) represents transformation of chronic lymphocytic leukemia (CLL) into a highly aggressive lymphoma with dismal prognosis. Transcriptomic alterations have been described in CLL but most studies focused on peripheral blood samples with minimal data on RS-involved tissue. Moreover, transcriptomic features of RS have not been well defined in the era of CLL novel therapies. In this study we investigated transcriptomic profiles of CLL/RS-involved nodal tissue using samples from a clinical trial cohort of refractory CLL and RS patients treated with Pembrolizumab (NCT02332980). Methods: Nodal samples from 9 RS and 4 CLL patients in MC1485 trial cohort were reviewed and classified as previously published (Ding et al, Blood 2017). All samples were collected prior to Pembrolizumab treatment. Targeted gene expression profiling of 789 immune-related genes were performed on FFPE nodal samples using Nanostring nCounter® Analysis System (NanoString Technologies, Seattle, WA). Differential expression analysis was performed using NanoStringDiff. Genes with 2 fold-change in expression with a false-discovery rate less than 5% were considered differentially expressed. Results: The details for the therapy history of this cohort were illustrated in Figure 1a. All patients exposed to prior ibrutinib before the tissue biopsy had developed clinical progression while receiving ibrutinib. Unsupervised hierarchical clustering using the 300 most variable genes in expression revealed two clusters: C1 and C2 (Figure 1b). C1 included 4 RS and 3 CLL treated with prior chemotherapy without prior ibrutinib, and 1 RS treated with prior ibrutinib. C2 included 1 CLL and 3 RS received prior ibrutinib, and 1 RS treated with chemotherapy. The segregation of gene expression profiles in samples was largely driven by recent exposure to ibrutinib. In C1 cluster (majority had no prior ibrutinb), RS and CLL samples were clearly separated into two subgroups (Figure 1b). In C2 cluster, CLL 8 treated with ibrutinib showed more similarity in gene expression to RS, than to other CLL samples treated with chemotherapy. In comparison of C2 to C1, we identified 71 differentially expressed genes, of which 34 genes were downregulated and 37 were upregulated in C2. Among the upregulated genes in C2 (majority had prior ibrutinib) are known immune modulating genes including LILRA6, FCGR3A, IL-10, CD163, CD14, IL-2RB (figure 1c). Downregulated genes in C2 are involved in B cell activation including CD40LG, CD22, CD79A, MS4A1 (CD20), and LTB, reflecting the expected biological effect of ibrutinib in reducing B cell activation. Among the 9 RS samples, we compared gene profiles between the two groups of RS with or without prior ibrutinib therapy. 38 downregulated genes and 10 upregulated genes were found in the 4 RS treated with ibrutinib in comparison with 5 RS treated with chemotherapy. The top upregulated genes in the ibrutinib-exposed group included PTHLH, S100A8, IGSF3, TERT, and PRKCB, while the downregulated genes in these samples included MS4A1, LTB and CD38 (figure 1d). In order to delineate the differences of RS vs CLL, we compared gene expression profiles between 5 RS samples and 3 CLL samples that were treated with only chemotherapy. RS samples showed significant upregulation of 129 genes and downregulation of 7 genes. Among the most significantly upregulated genes are multiple genes involved in monocyte and myeloid lineage regulation including TNFSF13, S100A9, FCN1, LGALS2, CD14, FCGR2A, SERPINA1, and LILRB3. Conclusion: Our study indicates that ibrutinib-resistant, RS-involved tissues are characterized by downregulation of genes in B cell activation, but with PRKCB and TERT upregulation. Furthermore, RS-involved nodal tissues display the increased expression of genes involved in myeloid/monocytic regulation in comparison with CLL-involved nodal tissues. These findings implicate that differential therapies for RS and CLL patients need to be adopted based on their prior therapy and gene expression signatures. Studies using large sample size will be needed to verify this hypothesis. Figure Disclosures Zhao: Merck: Current Employment. Blumenschein:Merck: Current Employment. Yearley:Merck: Current Employment. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Parikh:Verastem Oncology: Honoraria; GlaxoSmithKline: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; Merck: Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Kenderian:Sunesis: Research Funding; MorphoSys: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Gilead: Research Funding; BMS: Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Juno: Research Funding; Mettaforge: Patents & Royalties; Torque: Consultancy; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding. Kay:Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ding:DTRM: Research Funding; Astra Zeneca: Research Funding; Abbvie: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Secreted Factors Determine Resistance to Idelalisib in Marginal Zone Lymphoma Models of Resistance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2569-2569
Author(s):  
Alberto J Arribas ◽  
Sara Napoli ◽  
Eugenio Gaudio ◽  
Luciano Cascione ◽  
Alessandra Di Veroli ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Board ◽  
Institutional Research ◽  
Rna Seq ◽  
Scientific Advisory Board ◽  
Scientific Advisory ◽  
Erbb Signaling ◽  
Travel Grant

Background . PI3Kδ is expressed in B-cells and has a central role in the B-cell receptor signaling in B-cell derived malignancies. Idelalisib was the first-in-class PI3Kδ inhibitors and several second-generation compounds are undergoing clinical investigation as single agents and in combinations. To identify modalities to overcome the resistance that develops to this class of agents, we have developed two idelalisib-resistant models derived from splenic marginal zone lymphoma (SMZL) cell lines. Materials and Methods. Cells were kept under idelalisib (IC90) until acquisition of resistance (RES) or with no drug (parental, PAR). Stable resistance was confirmed by MTT assay after 2-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out. Cells underwent transcriptome and miRNA profiling by RNA-Seq, whole exome sequencing (WES), lipidomics profiling, pharmacological screening (348 compounds), and FACS analysis. Cytokines and growth factor secretion was performed by ELISA. Results. Two RES models were obtained from VL51 and Karpas1718 with 7-10 fold times higher IC50s than PAR counterparts. In both models, conditioned media from RES cells transferred the resistance in the PAR cells. While WES did not identify somatic mutations associated with resistance, RNA-Seq and lipidomics analyses showed that the two cell lines had developed resistance activating different modalities. The VL51 RES model showed an enrichment in BCR-TLR-NFkB (TLR4, CD19, SYK), IL6-STAT3 (IL6, CD44), chemokines (CXCL10, CXCR4, CXCR3) and PDGFR (PDGFRA, PRKCE) signatures, paired with increased p-AKT and p-BTK levels, decreased cardiolipins and sphingomyelins levels, and increased levels of specific triacylglycerols and glycerophosphocholines. In particular, there was an over-expression of surface expression of PDGFRA and secretion of IL6 in the medium. Silencing of both IL6and PDGFRA by siRNAs reverted the resistance, while the silencing of the individual genes had only a partial effect. These data were paired with the acquired sensitivity to the PDGFR inhibitor masitinib, identified in the pharmacologic screening. In the Karpas1718 model, we observed an increased p-AKT activity with an enrichment for B-cell activation signatures (RAG1, RAG2, TCL1A), proliferation (E2F2, MKI67), ERBB signaling (HBEGF, NRG2, ERRB4), increased levels of some triacylglycerols and repressed levels for specific glycerophosphocholines. HBEGF secretion was confirmed by ELISA. The addition of recombinant HBEGF to the medium induced resistance in the PAR cells. Combination with the pan ERBB inhibitor lapatinib was beneficial in the K1718 RES. Recombinant HBEGF also induced resistance to the BTK inhibitor ibrutinib in the PAR cells and in the mantle cell lymphoma SP-53 cell line. Specific members of the let-7 family of miRNAs were repressed in the RES lines derived from both cell lines, indicating the involvement of miRNA deregulation in the mechanism of resistance. Indeed, let-7 members are known to directly target IL6-STAT3 and cytokine signaling cascade, as well PI3K-AKT network. In solid tumors, let-7 members are also expressed at low levels in tumors with constitutive active ERBB signaling, in accordance with the activation of ERBB pathway and p-AKT we observed in our Karpas1718model. Experiments with a LIN28B inhibitor are now on-going. Finally, we validated the findings across a panel of 34 B-cell lymphoma cell lines, in which IL6, PDGFRA, HBEGF and LIN28 expression levels were negatively correlated with idelalisib sensitivity, while the latter was positively correlated with let-7 levels (P <0.05). Conclusions. We developed two distinct models derived from MZL of secondary resistance to the PI3Kδ inhibitor idelalisib. We identified treatments that might overcome resistance to idelalisib and are worth of further investigations. The two models, driven by different biologic processes, will allow the evaluation of further alternative therapeutic approaches. Disclosures Stathis: PharmaMar: Other: Renumeration; ADC Therapeutics: Other: Institutional research funding; Abbvie: Other: Renumeration; Bayer: Other: Institutional research funding; Novartis: Other: Institutional research funding; MEI-Pharma: Other: Institutional research funding; Roche: Other: Institutional research funding; Pfizer: Other: Institutional research funding; Merck: Other: Institutional research funding. Stuessi:Gilead: Speakers Bureau. Zucca:Gilead: Honoraria, Other: travel grant. Rossi:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board. Bertoni:Nordic Nanovector ASA: Research Funding; Acerta: Research Funding; Jazz Pharmaceuticals: Other: travel grants; ADC Therapeutics: Research Funding; Bayer AG: Research Funding; Cellestia: Research Funding; CTI Life Sciences: Research Funding; EMD Serono: Research Funding; Helsinn: Consultancy, Research Funding; ImmunoGen: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Oncology Therapeutic Development: Research Funding; PIQUR Therapeutics AG: Other: travel grant, Research Funding; HTG: Other: Expert Statements ; Amgen: Other: travel grants; Astra Zeneca: Other: travel grants.

scholarly journals Duohexabody-CD37, a Novel Bispecific Antibody with a Hexamerization-Enhancing Mutation Targeting CD37, Demonstrates Superior Complement-Dependent Cytotoxicity in Preclinical B-Cell Malignancy Models

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4170-4170
Author(s):  
Simone C. Oostindie ◽  
Hilma J. Van Der Horst ◽  
Marije B. Overdijk ◽  
Kristin Strumane ◽  
Sandra Verploegen ◽  
...  
Keyword(s):  
B Cell ◽  
Research Funding ◽  
Bispecific Antibody ◽  
Lymphocytic Leukemia ◽  
Single Point Mutation ◽  
B Cell Malignancies ◽  
Employment Equity ◽  
Healthy Human ◽  
Complement Dependent Cytotoxicity ◽  
Equity Ownership

Abstract CD37 is a tetraspanin plasma membrane protein abundantly expressed on B-cells and represents a promising therapeutic target for the treatment of B-cell malignancies. Although complement-dependent cytotoxicity (CDC) has proven to be a powerful Fc-mediated effector function for killing hematological cancer cells, CD37 antibody-based therapeutics currently in clinical development are poor inducers of CDC. Here we present DuoHexaBody-CD37, a novel humanized IgG1 bispecific antibody targeting two different CD37 epitopes, with an E430G hexamerization-enhancing mutation, for the potential treatment of B-cell malignancies. The natural process of antibody hexamer formation through intermolecular Fc-Fc interactions between IgG molecules after cell surface antigen binding can be improved by introducing a single point mutation such as E430G in the IgG Fc domain, thereby facilitating more efficient C1q binding and complement activation (Diebolder et al., Science 2014; de Jong et al., PLoS Biol 2016). The hexamerization-enhancing mutation E430G was introduced into two humanized CD37 monoclonal antibodies (mAbs) that bind non-overlapping CD37 epitopes. Different antibody formats and combinations, including the single antibodies, combinations of the mAbs and bispecific mAbs were tested for their capacity to induce CDC and antibody-dependent cellular cytotoxicity (ADCC). The bispecific hexamerization-enhanced antibody variant DuoHexaBody-CD37, showed superior CDC activity compared to the single hexamerization-enhanced mAbs and the combination thereof, both in vitro over a range of different B-cell lines, and ex vivo in tumor cell samples obtained from patients with chronic lymphocytic leukemia (CLL). In a CDC assay using tumor cells obtained from a relapsed/refractory CLL patient who received prior treatment with rituximab, ibrutinib and idelalisib, DuoHexaBody-CD37 induced almost complete lysis (84% lysis at a concentration 100 µg/mL), thereby outperforming the single HexaBody molecules (15% and 23% lysis) and the combination (57%) (Figure 1). In addition to its potent CDC activity, DuoHexaBody-CD37 was also capable of inducing potent ADCC of Daudi cells (EC50 = 12.3 ± 9.5 ng/mL), as assessed using peripheral blood mononuclear cells from 8 healthy human donors in a standard chromium release assay. In assays using whole blood from 6 healthy human donors, DuoHexaBody-CD37 showed efficient B-cell binding and potent and specific depletion of the B-cell population (98% ± 1.3% depletion at 10 µg/mL, EC50 = 0.85 ± 0.284 µg/mL). Furthermore, DuoHexaBody-CD37 induced significant inhibition of tumor growth in vivo in Daudi-luc Burkitt's lymphoma and JVM-3 CLL mouse xenograft models, at doses as low as 0.1 and 1 mg/kg (p<0.05), respectively. In summary, we present a novel therapeutic antibody that, for the first time, combines proprietary DuoBody® and HexaBody® platforms. DuoHexaBody-CD37 induced highly potent CDC and efficient ADCC in preclinical models, suggesting that DuoHexaBody-CD37 may serve as a potential therapeutic mAb for the treatment of human B-cell malignancies. Disclosures Oostindie: Genmab: Employment, Equity Ownership. Van Der Horst:Genmab: Research Funding. Overdijk:Genmab: Employment, Equity Ownership. Strumane:Genmab: Employment, Equity Ownership. Verploegen:Genmab: Employment, Equity Ownership. Lindorfer:Genmab: Research Funding. Cook:Genmab: Research Funding. Chamuleau:Gilead: Research Funding; BMS: Research Funding; celgene: Research Funding; Genmab: Research Funding. Mutis:Gilead: Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding; Novartis: Research Funding; OnkImmune: Research Funding. Schuurman:Genmab: Employment, Other: Warrants. Sasser:Genmab: Employment, Equity Ownership. Taylor:Genmab: Research Funding. Parren:Genmab: Equity Ownership; Lava Therapeutics: Employment. Beurskens:Genmab: Employment, Equity Ownership. Breij:Genmab: Employment, Equity Ownership.

scholarly journals The Broad Spectrum of TP53 Variants in CLL: NGS Analysis of 573 Pathogenic TP53 Variants

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3021-3021
Author(s):  
Gregory Lazarian ◽  
Floriane Theves ◽  
Myriam Hormi ◽  
Virginie Eclache ◽  
Stéphanie Poulain ◽  
...  
Keyword(s):  
Tumor Progression ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Fish Analysis ◽  
Snp Analysis ◽  
Advisory Committees ◽  
Exon 2 ◽  
Tp53 Mutations ◽  
Mutational Status

TP53 aberrations, including somatic mutations of TP53 gene or 17p deletion leading to the loss of the TP53 locus, are a major predictive factor of resistance to fludarabin based chemotherapy in chronic lymphocytic leukemia (CLL) and remain an adverse prognostic factor in the chemofree era. Therefore, detection of TP53 alteration before each new line of treatment is required for theranostic stratification. In order to better characterize the distribution and combination of the TP53 variants in CLL, we collected the TP53 sequencing data of 343 patients harboring TP53 mutations from centers of the French Innovative Leukemia Organization-CLL (FILO) and established a large data base of 573 TP53 mutations. Mutations were identified through NGS sequencing (covering exon 2 to 11) allowing the detection of low frequency variants down to 1% VAF. Several distinct low VAF mutations were orthogonally confirmed by digital PCR. TP53 variants were analyzed through UMD_TP53 data gathering 90 000 TP53 mutations from all type of cancers. IGHV mutational status and FISH analysis were available for 224 and 176 patients respectively. Using ACMG criteria from the UMD_TP53 database, we confirmed that 523 could be classified as pathogenic, 42 were likely pathogenic and 8 were VUS (Variants of Unknown Significance). As expected, the mutation distribution along the p53 protein exhibited a clustering of variants in the DNA binding domain of the protein. We also confirmed the presence of a specific hotspot at codon 234 (6%) which is noticeable in other CLL cohorts but absent in solid tumors. 431 TP53 variants led to the expression of a mutant protein whereas the remaining 142 led a TP53 null phenotype. For 8 patients without 17p deletion and a mutation VAF larger than 50%, SNP analysis indicate that these tumors had a copy number neutral loss of heterozygosis at 17p with a duplication of the mutant allele leading to homozygous mutations of TP53. When focusing on the allele burden of TP53 mutations, 264/573 (46%) variants had an allele frequency <10%. Even if they were predominantly found in polymutated cases, presence of only low VAF (<10%) mutations was evidenced in 74 (21%) patients (50 patients with a single TP53 mutation and 24 patients with more than one). All these cases would have been missed by conventional sequencing. Among the 343 patients, 113 (33%) were poly-mutated and harbored more than one pathogenic TP53 variants (2 to 11 variants per patient): 57 (16,7 %) had 2 variants, 32 (9,3%) had 3, 10 had 4 (3%) and 14 patients (4%) had 5 to 11 variants. Using both long range sequencing and in silico analysis, we could show that all these variants were distributed in different alleles supporting an important intratumoral heterogeneity and a strong selection for TP53 loss of function during tumor progression in these patients. Null variants were rarely found as single alteration: only 46 patients (13,4%) patients harbored a single null mutation. Null mutations were predominantly found in patients with multiclonal mutations (87% with 4 or more). Median size of variants significantly decreased with the number of mutations and most of low VAF (less than 10%) variants were found in multiclonal combinations. Multiclonal mutations were predominantly found in previously treated patients (41% treated versus 10 % untreated) but whether all these variants preceded treatment and were further selected is currently unknown. We observed that 71,5 % of patients were IGHV unmutated and multiclonal mutations were surprisingly more frequent in mutated IGHV cases than in unmutated ones. Only 50% of cases carried a 17p deletion, highlighting again the importance of testing for TP53 mutations in addition to FISH analysis. Presence or absence of 17p deletion was unrelated to the number of TP53 mutations. Taken together these observations suggest that the TP53 mutational landscape in CLL is very complex and can involve multiple mechanisms, converging to a total loss of TP53 function and tumor progression. NGS provides a powerful tool for detecting all these alterations including variants with low VAF and should become a standard for CLL screening prior to each line of treatment. Disclosures Leblond: Amgen: Honoraria, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Astra Zeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Letestu:Abbvie: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Roche: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Alexion: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts. Cymbalista:Abbvie: Honoraria; Roche: Research Funding; Sunesis: Research Funding; Gilead: Honoraria; Janssen: Honoraria; AstraZeneca: Honoraria.

Whole-exome and whole-genome sequencing in chronic lymphocytic leukemia: new biomarkers to target

2020 ◽  
Vol 21 (13) ◽  
pp. 957-962
Author(s):  
Charbel Hobeika ◽  
Gaelle Rached ◽  
Alain Chebly ◽  
Eliane Chouery ◽  
Hampig Raphael Kourie
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Throughput Sequencing ◽  
Gene Mutations ◽  
Lymphocytic Leukemia ◽  
Whole Genome ◽  
Mutational Status ◽  
Whole Exome ◽  
Worse Prognosis

Many biomarkers indicate prognosis in chronic lymphocytic leukemia; such as fluorescence in situ hybridization testing: 17p or 11q deletions have a worse prognosis than trisomy 12, 13q deletion or normal result, or the mutational status of the immunoglobulin heavy chain (IGHV): unmutated IGHV have a worse prognosis than mutated IGHV. Recently, many gene mutations ( TP53, NOTCH1 etc.,) have been linked to a worse prognosis. With the new era of high-throughput sequencing, it has become easier to study gene mutations and their implication in predicting prognosis. In this review, we aim to review all the studies that performed whole-exome sequencing or whole-genome sequencing on chronic lymphocytic leukemia cells and explore the implication of various genes in disease prognosis.

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